Kinase receptor activation assay

Abstract

An assay for measuring activation (i.e., autophosphorylation) of a tyrosine kinase receptor of interest is disclosed.(a) A first solid phase is coated with a substantially homogeneous population of cells so that the cells adhere to the first solid phase. The cells have either an endogenous tyrosine kinase receptor or have been transformed with DNA encoding a receptor or "receptor construct" and the DNA has been expressed so that the receptor or receptor construct is presented in the cell membranes of the cells.(b) A ligand is then added to the solid phase having the adhering cells, such that the tyrosine kinase receptor is exposed to the ligand.(c) Following exposure to the ligand, the adherent cells are solubilized, thereby releasing cell lysate.(d) A second solid phase is coated with a capture agent which binds specifically to the tyrosine kinase receptor, or, in the case of a receptor construct, to the flag polypeptide.(e) The cell lysate obtained in step (c) is added to the wells containing the adhering capture agent so as to capture the receptor or receptor construct to the wells.(f) A washing step is then carried out, so as to remove unbound cell lysate, leaving the captured receptor or receptor construct.(g) The captured receptor or receptor construct is exposed to a labelled anti-phosphotyrosine antibody which identifies phosphorylated residues in the tyrosine kinase receptor.(h) Binding of the anti-phosphotyrosine antibody to the captured receptor or receptor construct is measured.

What is claimed is:  
     
       1. A method for measuring phosphorylation of a kinase polypeptide comprising the steps of: 
       (a) coating a first solid phase with a homogeneous population of eukaryotic cells so that the cells adhere to the first solid phase, wherein the cells comprise a kinase polypeptide.  
       (b) exposing the adhering cells to an analyte;  
       (c) solubilizing the adhering cells, thereby releasing cell lysate therefrom;  
       (d) coating a second solid phase with a capture agent which binds specifically to the kinase polypeptide so that the capture agent adheres to the second solid phase;  
       (e) exposing the adhering capture agent to the cell lysate obtained in step (c) so that the kinase polypeptide adheres to the second solid phase;  
       (f) washing the second solid phase so as to remove unbound cell lysate;  
       (g) exposing the adhering kinase polypeptide to an antibody which identifies phosphorylated residues in the kinase polypeptide; and  
       (h) measuring binding of the antibody to the adhering kinase polypeptide, wherein the amount of antibody binding to the adhering kinase polypeptide is proportional to the amount of phosphorylation of said kinase polypeptide.  
     
     
       2. The method of claim  1 , wherein the cells are transformed with nucleic acid encoding the kinase polypeptide prior to step (a). 
     
     
       3. The method of claim  1 , wherein said kinase polypeptide is a tyrosine kinase polypeptide. 
     
     
       4. The method of claim  1 , wherein said kinase polypeptide is a serine kinase polypeptide. 
     
     
       5. The method of claim  1 , wherein said antibody identifies phosphorylated tyrosine residues in the kinase polypeptide. 
     
     
       6. The method of claim  1 , wherein said antibody identifies phosphorylated serine residues in the kinase polypeptide. 
     
     
       7. The method of claim  1 , wherein the analyte comprises a compound to activate the phosphorylation of the kinase polypeptide. 
     
     
       8. The method of claim  1 , wherein the cells comprise a mammalian cell line. 
     
     
       9. The method of claim  1 , wherein the cells are adherent. 
     
     
       10. The method of claim  1 , wherein the capture agent comprises a capture antibody. 
     
     
       11. The method of claim  1 , wherein the first solid phase comprises a well of a first assay plate. 
     
     
       12. The method of claim  11 , wherein the first assay plate is a cell culture assay plate. 
     
     
       13. The method of claim  11 , wherein between about 1×10 4  to 3×10 5  cells are added to the well in step (a). 
     
     
       14. The method of claim  1 , wherein the second solid phase comprises a well of a second assay plate. 
     
     
       15. The method of claim  1 , wherein the cell lysate is not concentrated or clarified prior to step (e). 
     
     
       16. The method of claim  11 , wherein step (c) comprises adding a lysis buffer to the well of the first assay plate and gently agitating the first assay plate. 
     
     
       17. The method of claim  16 , wherein the lysis buffer comprises a solubilizing detergent. 
     
     
       18. The method of claim  1 , wherein said antibody is labeled. 
     
     
       19. The method of claim  18 , wherein the label comprises an enzyme which is exposed to a color reagent and the color change of the color reagent is determined in (h). 
     
     
       20. The method of claim  1 , wherein said kinase polypeptide is an intracellular kinase polypeptide. 
     
     
       21. The method of claim  1 , wherein a block buffer is added to the second solid phase following step (d). 
     
     
       22. The method of claim  1 , wherein said kinase polypeptide comprises a flag polypeptide. 
     
     
       23. The method of claim  22 , wherein the flag polypeptide is fused to the carboxyl terminus of the kinase polypeptide.